Vitamin A (retinol) is a fat-soluble vitamin found mainly in fish liver oils, liver, egg yolk, butter, and cream. Green leafy and yellow vegetables contain beta-carotene and other provitamin carotenoids which are converted to retinol in the mucosal cells of the small intestine. Retinol cannot be synthesized in vivo and must be obtained from the diet. Retinol is metabolized into the biologically active derivative retinoic acid (RA) in a variety of cells. The 11-cis isomer of retinal (vitamin A.sub.1 aldehyde), combined with a protein moiety, forms the prosthetic group of photoreceptor pigments in the retina that are involved in night, day, and color vision. Retinol, RA, and other retinoids also influence epithelial cell differentiation.
A number of carrier proteins which bind retinol or other retinoids have been identified. These carrier proteins are similar to the fatty acid binding proteins, a family of small (14-16 kDa) cytosolic proteins which bind long-chain fatty acids, fatty acyl-coenzyme A (CoA) derivatives, and other hydrophobic molecules (Veerkamp, J. H. et al. (1991) Biochim. Biophys. Acta 108:1-24). Two intracellular proteins, termed cellular retinol-binding protein (CRBP) and cellular retinol-binding protein type II (CRBP II), are found within cells which participate in vitamin A metabolism or function. CRBP, which is expressed in numerous organs and tissues, delivers retinol to specific metabolic enzymes and to specific binding sites within the nucleus, and participates in the transepithelial movement of retinol across blood-organ barriers. CRBP II, which is expressed primarily in the small intestine, appears to be involved in the intestinal absorption of vitamin A (Ong, D. E. (1987) Arch. Dermatol. 123:1693-1695A).
Retinoid binding proteins appear to direct bound retinoid molecules to specific metabolic pathways. The retinoid binding proteins also protect the cell from the damaging effects of unliganded retinols (such as membrane disruption) and likewise protect structurally unstable retinols from non-enzymatic side reactions (such as isomerization and oxidation). Retinoid binding proteins also appear to function as sensors of retinoid concentration and act as modulators of retinoid metabolism (Napoli, J. L. (1996) FASEB J. 10:993-1001). Retinoid binding proteins have been cloned and characterized from a variety of sources including CRBP from Norway rat and from human (Levin, M. S. et al. (1987) J. Biol. Chem. 262: 7118-7124; Colantuoni, V. et al. (1985) Biochem. Biophys. Res. Commun. 130:431-439), and CRBP II from pig and from human (Perozzi, G. et al. (1993) J. Nutr. Biochem. 4:699-705; Loughney, A. D. et al. (1995) Hum. Reprod. 10:1297-1304).
X-ray crystallography of rat CRBP reveals a globular, flattened protein consisting of ten antiparallel strands of beta-sheet (Newcomer, M. E. (1995) FASEB J. 9:229-239). The retinol ligand is enclosed between two orthogonal sets of beta sheets, and the alcohol functional group is buried near the center of the protein and inaccessible to the cellular environment. The structure of the exterior surface of CRBP may direct the physiological interaction and transfer of bound retinols to specific retinoid metabolic enzymes and impart enzyme specificity to the retinoid metabolic pathways (Napoli, supra).
Since retinoids induce differentiation in immature hematopoietic and epithelial cell types, they are potential anti-cancer agents. A promyelocyte cell line was induced to differentiate to morphologically and functionally mature granulocytes by incubation with retinoic acid. Other retinoids, including retinol, retinal, and retinyl acetate, also induced differentiation, but higher concentrations were required (Breitman, T. R. et al. (1980) Proc. Natl. Acad. Sci. USA 77:2936-2940). Retinol is known to affect the differentiation of cultured keratinocytes derived from epidermis and other stratified squamous epithelia (Fuchs, E. et al. (1981) Cell 25:617-625). Retinoids and carotenoids have been proposed to have preventative and/or therapeutic effects on lung cancer and cardiovascular disease (Omenn, G. S. et al. (1996) N. Engl. J. Med. 334:1150-1155).
Retinoids have been found to be effective in suppressing tumor development in several carcinogenesis model systems and in human subjects (Lotan, R. (1996) FASEB J. 10:1031-1039, and references therein). Some retinoids were found to be active in certain animal models and not in others. The effect of retinoids was not restricted to a specific carcinogen, but rather to the type of tissue involved. This restriction suggests that some retinoids exhibit tissue selectivity. Other studies have demonstrated that certain retinoids which are active inhibitors of carcinogenesis in particular tissues can act as enhancers of carcinogenesis in the same tissue of a different strain of mouse or in another carcinogenesis model (Lotan, supra).
The abundance of intracellular retinoid binding proteins may have a role in the response of various tissues to retinoids. Treatment of F9 teratocarcinoma stem cells with retinoic acid (RA) causes their irreversible differentiation into extraembryonic endoderm. Boylan, J. F. et al. (1991; J. Cell. Biol. 112: 965-979) generated stably transfected F9 stem cell lines expressing either elevated or reduced levels of functional CRABP-I protein. CRABP-I is a retinol binding protein which preferentially binds retinoic acid. Cell lines expressing elevated levels of CRABP-I exhibited a significant reduction in the expression of RA-inducible mRNAs at low exogenous RA concentrations, but this reduction was eliminated at higher RA concentrations. Thus, higher levels of CRABP-I reduced the potency of RA in this differentiation system. Boylan et al. (supra) proposed that CRABP-I sequesters RA within the cell and thereby prevents RA from acting to regulate differentiation specific gene expression, and that the level of CRABP-I may affect tissue response to RA during development.
Retinoids affect sebaceous gland activity and exhibit immunomodulatory and anti-inflammatory properties (Orfanos, C. E. et al. (1997) Drugs 53:358-388). Retinoids have been used for topical and systemic treatment of psoriasis and other hyperkeratotic and parakeratotic skin disorders, severe acne and acne-related dermatoses, and for therapy and/or chemoprevention of skin cancer and other neoplasia including T-cell lymphoma (Orfanos et al., supra). Treatment of human pancreatic carcinoma cell lines with retinoids resulted in growth inhibition and differentiation of ductal, but not acinar, pancreatic tumor cells (Rosewicz, S. et al. (1995) Gastroenterology 109:1646-1660). CRABP II was found in all retinoid-sensitive ductal tumor cell lines but not in the retinoid-resistant acinar cell lines.
Toxic side-effects associated with retinoid treatments include changes in the skin and mucous membranes (dry skin, hair loss, dry nose, conjunctivitis), musculoskeletal symptoms, ophthalmological effects, changes in transaminase activity, changes in clinical chemistry markers (increase in serum triglycerides and decrease in high-density lipoproteins), and, rarely, central nervous system effects. Most of the side-effects are reversed after stopping treatments. A serious toxicological aspect of retinoid treatment is teratogenesis. Retinoid therapies are thus not recommended for women of childbearing age, and conception should be prevented for a significant period of time after stopping treatment (La Vecchia, C. et al. (1996) IARC Sci. Publ. 139:135-142).
Inadequate intake or utilization of vitamin A can impair dark adaptation and cause night blindness; xerosis of the conjunctiva and cornea; xerophthalmia and keratomalacia; keratinization of lung, GI tract, and urinary tract epithelia; and increased susceptibility to infections. Defective taste and smell, and anemia that may be masked by hemoconcentration, have also been reported (Berkow, R. and Fletcher, A. J., eds. (1992) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J., pp. 959-960).
Primary vitamin A deficiency is usually caused by prolonged dietary deprivation. It is endemic in areas such as southern and eastern Asia where rice, devoid of carotene, is the staple. Secondary deficiency may be due to inadequate conversion of carotene, or to interference with absorption, storage, or transport of vitamin A. Interference with absorption or storage is likely in celiac disease, sprue, cystic fibrosis, operations on the pancreas, duodenal bypass, congenital partial obstruction of the jejunum, obstruction of the bile ducts, giardiasis, and cirrhosis of the liver. Vitamin A deficiency is common in protein-energy malnutrition, not only because the diet is deficient, but because vitamin A storage and transport are defective. Liver stores are depleted in deficiency before plasma levels begin to fall, followed later by retinal dysfunction, and finally by epithelial structural changes.
Discovery of a new human retinoid binding protein and the polynucleotides which encode it satisfies a need in the art by providing new compositions useful in diagnosing and treating disorders associated with cellular development and differentiation and retinoid metabolism.